Skip to main content
SpringerLink
Log in

Comprehensive Profiling of Proteome Changes Provide Insights of Industrial Penicillium chrysogenum During Pilot and Industrial Penicillin G Fermentation

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The intracellular proteomes of the Penicillium chrysogenum throughout pilot and industrial processes were investigated by using 2-DE combined with MALDI-TOF-TOF MS, respectively. We detected a total of 223 spots corresponding to 154 proteins and 231 spots corresponding to 157 proteins throughout pilot and industrial processes, respectively. The levels of glyceraldehyde-3-phosphate dehydrogenase increased (5.1- and 2.5-fold) under the pilot process, while its levels were no significant changes under the industrial process at 140 and 170 h when compared with that at 2 h. The levels of isocitrate lyase and fumarate hydratase were increased significantly under the industrial process, while their levels had no obvious changes after 20 h of fermentation throughout the pilot process. These results indicate that there were remarkable differences in carbohydrate metabolism (including glycolysis, gluconeogenesis, pentose phosphate pathway, and tricarboxylic acid cycle) of P. chrysogenum during the pilot and industrial fermentations, which likely result in alterations of the primary metabolism and penicillin biosynthesis. Moreover, the differences in the levels of proteins involved in amino acid metabolisms (including valine, cysteine, and α-aminoadipic acid biosynthesis) indicated that the pilot and industrial processes influenced the supplies of penicillin precursors. Compared with that at 2 h, the maximum levels of superoxide (6.9-fold, at 32 h) and catalase (9-fold, at 80 h) during the industrial process and the maximum levels of superoxide (1.2-fold, at 20 h) and catalase (7.7-fold at 128 h) during the pilot process revealed the significant difference in cell redox homeostasis and stress responses during scale-up fermentation. Particularly, 10 spots corresponding to isopenicillin N synthetase and 4 spots corresponding to isopenicillin N (IPN) acyltransferase in pilot and industrial processes were identified, respectively. The levels of IPN acyltransferase (spots 197 and 198) and CoA ligase at 80 h during the industrial process were around 2-fold of that during the pilot process, indicating that the industrial process with a higher penicillin production per cell might provide available environments to induce over-expression of IPN acyltransferase and accelerate penicillin formation. These results provide new insights into the globally potential responses of P. chrysogenum to variations of environments in different fermentation scales so as to consequently regulate the penicillin production.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. van den Berg, M. A., Albang, R., Albermann, K., Badger, J. H., Daran, J. M., Driessen, A. J., Garcia-Estrada, C., Fedorova, N. D., Harris, D. M., Heijne, W. H., Joardar, V., Kiel, J. A., Kovalchuk, A., Martín, J. F., Nierman, W. C., Nijland, J. G., Pronk, J. T., Roubos, J. A., van der Klei, I. J., van Peij, N. N., Veenhuis, M., von Döhren, H., Wagner, C., Wortman, J., & Bovenberg, R. A. (2008). Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nature Biotechnology, 26, 1161–1168.

    Article  CAS  Google Scholar 

  2. Peñalva, M. A., Rowlands, R. T., & Turner, G. (1998). The optimization of penicillin biosynthesis in fungi. Trends in Biotechnology, 16, 483–489.

    Article  Google Scholar 

  3. Thykaer, J., & Nielsen, J. (2003). Metabolic engineering of beta-lactam production. Metabolic Engineering, 5, 56–69.

    Article  CAS  Google Scholar 

  4. Dantigny, P., & Nanguy, S. P. (2009). Significance of the physiological state of fungal spores. International Journal of Food Microbiology, 134, 16–20.

    Article  CAS  Google Scholar 

  5. van de Kamp, M., Driessen, A. J., & Konings, W. N. (1999). Compartmentalization and transport in beta-lactam antibiotic biosynthesis by filamentous fungi. Antonie Van Leeuwenhoek, 75, 41–78.

    Article  Google Scholar 

  6. Kiel, J. A., van den Berg, M. A., Fusetti, F., Poolman, B., Bovenberg, R. A., Veenhuis, M., & van der Klei, I. J. (2009). Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells. Functional & Integrative Genomics, 9, 167–184.

    Article  CAS  Google Scholar 

  7. Harris, D. M., van der Krogt, Z. A., Klaassen, P., Raamsdonk, L. M., Hage, S., van den Berg, M. A., Bovenberg, R. A., Pronk, J. T., & Daran, J. M. (2009). Exploring and dissecting genome-wide gene expression responses of Penicillium chrysogenum to phenylacetic acid consumption and penicillinG production. BMC Genomics, 10, 75.

    Article  Google Scholar 

  8. Castillo, N. I., Fierro, F., Gutiérrez, S., & Martín, J. F. (2006). Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source. Current Genetics, 49, 85–96.

    Article  CAS  Google Scholar 

  9. Brakhage, A. A., Spröte, P., Al-Abdallah, Q., Gehrke, A., Plattner, H., & Tüncher, A. (2004). Regulation of penicillin biosynthesis in filamentous fungi. Advances in Biochemical Engineering / Biotechnology, 88, 45–90.

    CAS  Google Scholar 

  10. Harris, D. M., Diderich, J. A., van der Krogt, Z. A., Luttik, M. A., Raamsdonk, L. M., Bovenberg, R. A., van Gulik, W. M., van Dijken, J. P., & Pronk, J. T. (2006). Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase. Metabolic Engineering, 8, 91–101.

    Article  CAS  Google Scholar 

  11. Zhao, Z., Kuijvenhoven, K., Ras, C., van Gulik, W. M., Heijnen, J. J., Verheijen, P. J., & van Winden, W. A. (2008). Isotopic non-stationary 13C gluconate tracer method for accurate determination of the pentose phosphate pathway split-ratio in Penicillium chrysogenum. Metabolic Engineering, 10, 178–186.

    Article  CAS  Google Scholar 

  12. Kleijn, R. J., Liu, F., van Winden, W. A., van Gulik, W. M., Ras, C., & Heijnen, J. J. (2007). Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. Metabolic Engineering, 9, 112–123.

    Article  CAS  Google Scholar 

  13. Nasution, U., van Gulik, W. M., Proell, A., van Winden, W. A., & Heijnen, J. J. (2006). Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor. Metabolic Engineering, 8, 395–405.

    Article  CAS  Google Scholar 

  14. Nasution, U., van Gulik, W. M., Ras, C., Proell, A., & Heijnen, J. J. (2008). A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum. Metabolic Engineering, 10, 10–23.

    Article  CAS  Google Scholar 

  15. Jami, M. S., Barreiro, C., García-Estrada, C., & Martín, J. F. (2010). Proteome analysis of the penicillin producer Penicillium chrysogenum: characterization of protein changes during the industrial strain improvement. Molecular & Cellular Proteomics, 9, 1182–1198.

  16. Thykaer, J., Rueksomtawin, K., Noorman, H., & Nielsen, J. (2008). NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production. Microbiology, 154, 1242–1250.

    Article  CAS  Google Scholar 

  17. Thykaer, J., Christensen, B., & Nielsen, J. (2000). Metabolic network analysis of an adipoyl-7-ADCA-producing strain of Penicillium chrysogenum: elucidation of adipate degradation. Metabolic Engineering, 4, 151–158.

    Article  Google Scholar 

  18. van Winden, W. A., van Gulik, W. M., Schipper, D., Verheijen, P. J., Krabben, P., Vinke, J. L., & Heijnen, J. J. (2003). Metabolic flux and metabolic network analysis of Penicillium chrysogenum using 2D [C-13, H-1] COSYNMR measurements and cumulative Bondomer simulation. Biotechnology and Bioengineering, 83, 75–92.

    Article  Google Scholar 

  19. Cao, Y. X., Qiao, B., Lu, H., Chen, Y., & Yuan, Y. J. (2011). Comparison of the secondary metabolites in Penicillium chrysogenum between pilot and industrial penicillin G fermentations. Applied Microbiology and Biotechnology, 89, 1193–1202.

    Article  CAS  Google Scholar 

  20. Cheng, J. S., Ding, M. Z., Tian, H. C., & Yuan, Y. J. (2009). Inoculation-density-dependent responses and pathway shifts in Saccharomyces cerevisiae. Proteomics, 9, 4704–4713.

    Article  CAS  Google Scholar 

  21. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  22. Shamir, R., Maron-Katz, A., Tanay, A., Linhart, C., Steinfeld, I., Sharan, R., Shiloh, Y., & Elkon, R. (2005). EXPANDER—an integrative program suite for microarray data analysis. BMC Bioinformatics, 6, 232.

    Article  Google Scholar 

  23. Ding, M. Z., Lu, H., Cheng, J. S., Chen, Y., Jiang, J., Qiao, B., Li, B. Z., & Yuan, Y. J. (2012). Comparative metabolomic study of Penicillium chrysogenum during pilot and industrial penicillin fermentations. Applied Biochemistry and Biotechnology, 168, 1223–1238.

    Article  CAS  Google Scholar 

  24. Pollard, D. J., Kirschner, T. F., Hernandez, D., Hunt, G., Olewinski, R., & Salmon, P. M. (2002). Pilot-scale process sensitivity studies for the scale up of a fungal fermentation for the production of pneumocandins. Biotechnology and Bioengineering, 78, 270–279.

    Article  CAS  Google Scholar 

  25. Douma, R. D., Verheijen, P. J., de Laat, W. T., Heijnen, J. J., & van Gulik, W. M. (2010). Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum. Biotechnology and Bioengineering, 106, 608–618.

    Article  CAS  Google Scholar 

  26. Naranjo, L., Martín de Valmaseda, E., Casqueiro, J., Ullán, R. V., Lamas-Maceiras, M., Bañuelos, O., & Martín, J. F. (2004). Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid. Applied and Environmental Microbiology, 70, 1031–1039.

    Article  CAS  Google Scholar 

  27. Esmahan, C., Alvarez, E., Montenegro, E., & Martin, J. F. (1994). Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis. Applied and Environmental Microbiology, 60, 1705–1710.

    CAS  Google Scholar 

  28. Emri, T., Pócsi, I., & Szentirmai, A. (1999). Analysis of the oxidative stress response of Penicillium chrysogenum to menadione. Free Radical Research, 30, 125–132.

    Article  CAS  Google Scholar 

  29. Wang, F. Q., Zheng, G. Z., Zhao, Y., Ren, Z. H., Jia, Q., He, J. G., & Yu, J. (2006). Molecular cloning and characterization of a glutathione S-transferase gene repressed by phenylacetic acid from Penicillium chrysogenum. Progress in Biochemistry and Biophysics, 33, 1223–1230.

    CAS  Google Scholar 

  30. Sámi, L., Karaffa, L., Emri, T., & Pócsi, I. (2003). Autolysis and ageing of Penicillium chrysogenum under carbon starvation: respiration and glucose oxidase production. Acta Microbiologica et Immunologica Hungarica, 50, 67–76.

    Article  Google Scholar 

  31. Koetsier, M. J., Jekel, P. A., van den Berg, M. A., Bovenberg, R. A., & Janssen, D. B. (2009). Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum. Biochemical Journal, 417, 467–476.

    Article  CAS  Google Scholar 

  32. Jüsten, P., Paul, G. C., Nienow, A. W., & Thomas, C. R. (1998). Dependence of Penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity. Biotechnology and Bioengineering, 59, 762–775.

    Article  Google Scholar 

  33. Nasution, U., van Gulik, W. M., Kleijn, R. J., van Winden, W. A., Proell, A., & Heijnen, J. J. (2006). Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated Penicillium chrysogenum. Biotechnology and Bioengineering, 94, 159–166.

    Article  CAS  Google Scholar 

  34. Jorgensen, H., Nielsen, J., Villadsen, J., & Mollgaard, H. (1995). Metabolic flux distributions in Penicillium chrysogenum during fed-batch cultivations. Biotechnology and Bioengineering, 46, 117–131.

    Article  CAS  Google Scholar 

  35. Guais, O., Borderies, G., Pichereaux, C., Maestracci, M., Neugnot, V., Rossignol, M., & François, J. M. (2008). Proteomics analysis of “Rovabiot Excel”, a secreted protein cocktail from the filamentous fungus Penicillium funiculosum grown under industrial process fermentation. Journal of Industrial Microbiology & Biotechnology, 35, 1659–1668.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful for the financial support from the National Basic Research Program of China (973 Program: 2014CB745100), the National Natural Science Foundation of China (Program: 21176183, 21576201), and the Natural Science Foundation of Tianjin (Key program: 11JCZDJC16700).

Author information

Authors and Affiliations

  1. Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin, 300072, People’s Republic of China

    Jing-Sheng Cheng, Yan Zhao, Bin Qiao, Hua Lu & Ying-Jin Yuan

  2. Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People’s Republic of China

    Jing-Sheng Cheng, Yan Zhao, Bin Qiao, Hua Lu & Ying-Jin Yuan

  3. Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, Tianjin, 300072, People’s Republic of China

    Jing-Sheng Cheng, Yan Zhao, Bin Qiao & Ying-Jin Yuan

  4. Hebei Zhongrun Pharmaceutical Co., Ltd., China, Shijiazhuang Pharmaceutical Group Co., Ltd. (CSPC), Shijiazhuang, Hebei Province, 050041, People’s Republic of China

    Hua Lu & Yao Chen

Authors
  1. Jing-Sheng Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yan Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bin Qiao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hua Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yao Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ying-Jin Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jing-Sheng Cheng, Hua Lu or Ying-Jin Yuan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 2596 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cheng, JS., Zhao, Y., Qiao, B. et al. Comprehensive Profiling of Proteome Changes Provide Insights of Industrial Penicillium chrysogenum During Pilot and Industrial Penicillin G Fermentation. Appl Biochem Biotechnol 179, 788–804 (2016). https://doi.org/10.1007/s12010-016-2031-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2031-x

Keywords

Search

Navigation